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In all but the simplest cells, a protein called actin organizes to support the cell’s shape. It typically does so in linear or branched formations. Turns out, actin can come together in a completely different way as well, according to work by HHMI investigator Xiaowei Zhuang and her lab team.

In the thin, extending part of a nerve cell, called the axon, actin forms rings, encircling the length of the fiber at regular intervals. Zhuang suggests that these repeated rings lend flexible yet robust support to axons. She also proposes that the pattern may affect how electric signals are transmitted along axons.

“No one has ever seen periodic rings like this formed by actin,” says Zhuang, a biophysicist at Harvard University whose team published the findings on January 25, 2013, in Science. The discovery comes as a surprise to neuroscientists, who have pondered the structure of actin within brain cells—in particular, within axons and the threadlike extensions of the nerve cell body known as dendrites.

At less than a ten-millionth of a millimeter (10 nanometers) in diameter, the protein was too small for most light microscopes to visualize in detail. Early last year, Zhuang’s postdoc Ke Xu improved a microscopic technique developed in Zhuang’s lab called STORM (stochastic optical reconstruction microscopy) and captured high-resolution images of individual filaments of actin in fibroblasts and epithelial cells—work published January 8, 2012, in Nature Methods. Soon after, Zhuang challenged Xu and another postdoc in the lab, Guisheng Zhong, to image actin within brain cells, where its structural organization is thought to influence brain cell communication and other neurologic processes.

To create an image with STORM (stochastic optical reconstruction microscopy), researchers label the molecules they want to study with fluorescent probes, and then use a burst of light to activate the fluorescence in a small percentage of the molecules. A microscope captures an image of the fluorescing probes. The process is repeated many times, capturing a different subset of molecules with each burst of light. A final compilation of the images shows each molecule in its precise location in the cell with nanometer accuracy. From Rust et al, Nature Methods 3: 793-796 (2006) and Huang et al, Cell 143: 1047-1058 (2010).

A conventional fluorescence microscopy image of actin protein in the dendrite of a neuron appears blurry due to limited resolution. From Xu et al, Science 339: 452-456 (2013).

In contrast, single actin filaments are clearly resolved in the STORM image. Long actin filaments can be seen aligning along the neuron’s dendritic shaft. From Xu et al, Science 339: 452-456 (2013).

Zhuang and her colleagues discovered the organization of actin in axons is dramatically different from that in dendrites. STORM images (bottom) revealed that actin formed isolated ring-like structures that were evenly spaced along axons. The structures were obscured in conventional fluorescence images (top). From Xu et al, Science 339: 452-456 (2013).

The researchers discovered that a protein called spectrin also forms periodic, ladder-like structures interspersed with actin in axons. From Xu et al, Science 339: 452-456 (2013).

Based on her team’s work, Zhuang believes that short actin filaments (green), capped on one end by a protein called adducin (blue), form ring-like structures that wrap around the circumference of the axon. Spectrin tetramers (pink) connect adjacent actin rings, creating the periodic lattice seen in STORM images. From Xu et al, Science 339: 452-456 (2013).

Zhuang suggested that Xu and Zhong focus on actin in the synapses—specialized cell–cell junctions responsible for communication—because neuroscientists predicted that the protein’s structure at these junctions would influence chemical signaling. Instead, the two became preoccupied with fluorescent dots that aligned along the neurons’ axonal fibers. Over the following months, Xu and Zhong perfected their methods of labeling and imaging neurons until they could image actin clearly in three dimensions. The images revealed that the dots were in fact individual rings of actin periodically encircling the axonal fibers just beneath the membrane.

In addition, they found that one of actin’s binding partners, a protein called spectrin, was also part of the pattern. Zhong and Xu fluorescently labeled spectrin and saw that a long stretch of the protein—180 to 190 nanometers—bridged each actin ring.

The team revealed that sodium ion channels embedded in the axonal membrane follow a periodic pattern as well. Ions passing through these channels create electric charges that rise and fall rapidly, sending signals throughout the brain as they travel along axons. This electric movement is detailed in a mathematical model called the cable equation. As a result of the team’s findings, “The outcome of the equation that describes how electrical signals propagate along axons may now need to be modified,” says Zhuang’s colleague, Harvard chemical biologist Xiaoliang “Sunney” Xie.

Zhuang credits her postdocs for having the intuition to pursue whether the “dots” they saw in STORM’s first image were more than an artifact and for having the endurance to enrich and perfect the results until they could construct a model of this molecular lattice. “When they first noticed the seemingly periodic pattern, it was not so obvious at all, but they did not let it go,” recalls Zhuang. “These talented guys were so persistent in overcoming one difficulty after another to optimize the experimental conditions and generate these convincing images.”

In these two iBioSeminars, Xiaowei Zhuang explains the STORM technique and gives examples of how her lab has used STORM.

Zhuang would like to capture even finer images of actin–spectrin interaction within the lattice and to better understand the functional role it serves. She plans to improve STORM so that it can delve down from 10 nanometers to just a few nanometers. A STORM microscope snaps only a subset of glowing fluorescent molecules in a sample at a time and then iterates this process until a high-resolution image is reconstructed. In contrast, conventional microscopes image all glowing molecules at once, creating a blur that limits resolution. To increase STORM’s power, Zhuang needs to find brighter fluorescent tags that label molecules of interest in the sample more densely and hone the microscope to determine the positions of the molecules more precisely.

Xie attributes Zhuang’s unexpected finding to her focus on perfecting techniques and exploring biologically important topics. She had the foresight to focus on actin in the brain, he says, with one of the only tools capable of doing it.

Unveiling the structure of the actin–spectrin rings speaks to the worth of super-resolution microscopes like STORM, Zhuang says. “Making a scientific discovery is extremely rewarding for people like us who develop new methods. It is the ultimate validation of a method."